Objective: To screen serum protein markers and evaluate their diagnostic application value in hepatitis B-related acute-on-chronic liver failure (HBV-ACLF). Methods: Serum samples of patients with HBV-ACLF, chronic hepatitis B (CHB) and normal healthy volunteers (n = 5/group) were determined by cytokine antibody chip in line with the Chinese Diagnostic Standards Study for HBV-ACLF (COSSH-ACLF) cohort. The differentially expressed proteins significance were identified by microarray analysis and prediction. The preliminary serological markers of HBV-ACLF were screened for diagnosis. The potential markers were determined by enzyme-linked immunosorbent assay (ELISA), area under the receiver operating characteristic curve (AUROC) analysis and liver tissue immunohistochemistry for the diagnosis of HBV-ACLF. Student t-test or Mann-Whitney U test were used to compare the continuous measurement data between the two groups, and analysis of variance and Kruskal-Wallis test were used to compare the continuous measurement data between multiple groups. Results: Cytokine antibody chip preliminary screening results showed that the expression levels of these six cytokines, namely, macrophage inflammatory protein 3α (MIP-3α), hepatocyte growth factor, E-selectin, osteopontin, growth differentiation factor 15 and carcinoembryonic antigen-related cellular adhesion molecule 1 were significantly increased in the HBV-ACLF group. Among them, the expression level of MIP-3α was significantly higher in the HBV-ACLF group (99.6 times higher than CHB group and 146.9 times higher than healthy volunteers' group, respectively, P < 0.0001) as validated by serum ELISA in 132 HBV-ACLF cases, 91 CHB cases and 72 healthy volunteers. AUROC analysis showed that the high expression of MIP-3α could be used as a marker to distinguish patients with HBV-ACLF from CHB. The AUROC was 0.995 (95% CI: 0.990 ~ 1.000), with sensitivity and specificity of 95.5% and. 98.9%, respectively. Immunohistochemistry showed that MIP-3α was positively expressed in HBV-ACLF-derived liver tissues, and negatively expressed in CHB-derived liver and normal liver tissues. Conclusion: Serum MIP-3α level is closely related to the pathological characteristics of HBV-ACLF. Therefore, it may be used as a potential serological marker for the diagnosis of HBV-ACLF.
目的： 筛选乙型肝炎相关的慢加急性肝衰竭（HBV-ACLF）血清蛋白标志物并评价其在诊断中的应用价值。 方法： 用HBV-ACLF中国诊断标准研究（COSSH-ACLF）队列，应用细胞因子抗体芯片对HBV-ACLF患者、慢性乙型肝炎（CHB）患者和正常健康志愿者（n = 5/组）的血清样本进行检测，通过微阵列显著性分析和微阵列预测分析法寻找差异表达蛋白，初步筛查诊断HBV-ACLF的血清学标志物。通过酶联免疫吸附法（ELISA）、受试者操作特征曲线下面积（AUROC）分析以及肝组织免疫组织化学等方法进行验证，发现诊断HBV-ACLF的潜在标志物。用Student t检验或Mann-Whitney U检验比较两组间的连续计量数据，用方差分析和Kruskal-Wallis检验比较多组间的连续计量数据。 结果： 通过细胞因子抗体芯片初始筛选，发现巨噬细胞炎性蛋白3α（MIP-3α）、肝细胞生长因子、E-选择素、骨桥蛋白、生长分化因子15和癌胚抗原相关细胞黏附分子1这6个细胞因子在HBV-ACLF组中的表达水平显著升高。其中，MIP-3α的表达水平经132例HBV-ACLF患者、91例CHB患者和72名正常健康志愿者血清ELISA验证，在HBV-ACLF组中显著高表达（分别为CHB组的99.6倍、正常对照组的146.9倍，P < 0.000 1）。AUROC分析显示，MIP-3α在HBV-ACLF患者中的高表达可作为区分HBV-ACLF和CHB的标志物，AUROC为0.995（95% CI：0.990~1.000），灵敏度和特异度分别为95.5%和98.9%。免疫组织化学显示MIP-3α在HBV-ACLF来源的肝组织中表达为阳性，在CHB来源的肝组织和正常肝组织中表达为阴性。 结论： 血清MIP-3α水平与HBV-ACLF病理特征密切相关，MIP-3α有潜力作为HBV-ACLF诊断的血清学标志物。.
Keywords: Chronic hepatitis B; Liver failure; Macrophage inflammatory protein 3α.